KIT is a 145 kD type III tyrosine kinase receptor that functions as a growth factor receptor. KIT ligand is the Stem Cell Factor (SCF) (Roskoski 2005). Type III tyrosine kinase receptors are characterized by the presence of five N-glycosylated immunoglobulin domains in the extra-cellular region and by a split tyrosine kinase intra-cellular domain. The first three extra-cellular domains (D1-3) are involved in the binding of SCF (Lev, Blechman et al. 1993) while the fourth (D4) and fifth (D5) domains are implicated in receptor dimerization following SCF binding (Blechman and Yarden 1995). The intracellular region of KIT contains the catalytical domain composed of the ATP binding site and the phosphotransferase domain. KIT stimulation by SCF induces its dimerization and autophosphorylation which activates downstream effector proteins including the phosphoinositide 3-kinase (PI3K)/AKT, phospholipase C, signal transducer and activator of transcription (STAT) and RAS/MAP-kinase pathways (Roskoski 2005). SCF is a major cytokine for the self-renewal, proliferation and differentiation of hematopoietic lineage, germ cells, melanocytes, gut and central nervous system in embryo (Orr-Urtreger, et al. 1990). In adult mice KIT is expressed in a limited number of tissues and KIT defects induce impaired hematopoïesis, decreased numbers of tissue mast cells, decreased fertility and pigmentation, and defective development of the interstitial cells of Cajal, which are responsible for intestinal pacemaker activity (for review see Broudy 1997).
Abnormal KIT signalling is observed in cancer due either to overexpression SCF and/or KIT itself or to activating mutations that render KIT signalling independent of SCF. These mutations of KIT are major oncogenic drivers in gastrointestinal stromal tumours (GIST) (Demetri, von Mehren et al. 2002), that derive from Cajal cells, in subsets of acute myeloid leukemia (Core-binding factor acute myeloid leukemia CBF-AML) (Wang, Zhao et al. 2011) and melanoma (Hodi, Corless et al. 2013) and less frequently, in other cancers. Consequently, oncogenic KIT inhibition with tyrosine kinase inhibitors has proven successful in those pathologies (for review, Lennartsson and Ronnstrand 2012). Un-mutated KIT, is also involved in a number of malignant diseases derived from cell-types that generally expressed KIT transiently during embryogenesis (Bernex, De Sepulveda et al. 1996). The SCF/KIT axis functions as an autocrine or paracrine loop sustaining cancer cells to proliferate and/or to migrate. Indeed, KIT is present on 50% of AML (Ikeda, Kanakura et al. 1991) and AML blasts frequently respond to SCF stimulation by increased proliferation (Pietsch et al., 1992). Consistently, cases of AML patients, refractory to chemotherapy, can be cured by TKI (Xiang, Kreisel et al. 2007). A number of solid tumours also frequently express KIT and/or SCF including small cell lung cancer (SCLC), melanoma, semimoma (Went, Dirnhofer et al. 2004). Typically, seventy % of SCLC co-express SCF and KIT (Hibi, Takahashi et al. 1991; Rygaard, Nakamura et al. 1993; Krystal, Hines et al. 1996) and high KIT levels are associated with a poor prognosis (Micke, Basrai et al. 2003) while KIT kinase inhibitor Imatinib decreases cancer cell proliferation in vitro (Krystal, Honsawek et al. 2000) and reduces SCF-dependent VEGF secretion (Litz and Krystal 2006). Small cell lung cancer (NSCLC), also frequently express KIT (Yoo, Kim et al. 2004) and interestingly, Imatinib or SCF-blocking mAbs eliminate NSCLC cancer stem cell sub-population (Levina, Marrangoni et al. 2010). Other solid tumours have been reported to express KIT including breast cancers (Hines et al., 1995), neuroblastomas (Cohen et al., 1994), colon cancers (Toyota et al., 1994), gynecological tumours (Inoue et al., 1994), and gliomas (Stanulla et al., 1995).
Pharmacologic inhibition of KIT is a potential approach to treat malignancies that are partly or completely dependent on the activity of KIT receptor. ATP-competitive tyrosine kinase inhibitors like Imatinib (Heinrich, Griffith et al. 2000) or Dasatinib (Schittenhelm, Aichele et al. 2003) inhibit KIT phosphorylation and interfere with cell growth or survival. Unfortunately, a number KIT mutations located in the activating loop exhibits resistance to Imatinib in the clinic (Zermati, De Sepulveda et al. 2003; Ma, Mali et al. 2012) and initially Imatinib sensitive regulatory mutants accumulate secondary mutations leading to patient resistance to treatment (Antonescu, Besmer et al. 2005).
Because of their high specificity and high binding affinity combined to their natural ability to recruit immune effectors, monoclonal antibodies (mAbs) (Adams and Weiner 2005) offer an attractive approach to target oncogenic KIT. Thus, recently an anti-KIT mAb (SR-1, initially disclosed in the International Patent Application WO92/17505) was reported to limit tumour progression by a macrophage-dependent mechanism in a mouse model of GIST (Edris, Willingham et al. 2013) and a radiolabelled anti-KIT antibody (12A8 mAb) was shown to decrease SCLC progression in mouse models (Yoshida, Tsuji et al. 2013). Antibody SR1 has been shown to inhibit SCF binding to KIT and SCF inhibits SR1 binding to KIT (Ashman, Buhring, et al. 1994). Additionally, monoclonal antibodies targeting Domain 4 of KIT have been described to neutralize KIT dependant cell growth by inhibition of homotypic D4 interactions essential for KIT activation (Reshetnyak, Nelson et al. 2013).
Recently, monoclonal antibodies that specifically target the extracellular domain (ECD) of human c-KIT have been disclosed (International patent application N° WO2012/154480). These monoclonal antibodies that specifically target the ECD of human c-KIT, including the CK6 antibody) are presented as capable of inducing internalization and/or degradation of plasma membrane bound c-KIT without inducing c-KIT agonist activities in tumor cells. However, the results disclosed in said patent application are in contradiction with this assertion since anti-c-KIT mAb CK6 is not observed as significantly inducing degradation of c-KIT expressed by GIST882 human tumor cells, expressed by Mo7e human tumor cells and expressed by Malm-3M human tumor cells in a time-dependent manner.
Therefore, until now, no monoclonal antibodies capable of inducing internalization and degradation of oncogenic forms of KIT have been described. Moreover, no neutralizing antibody binding to D5 of KIT has been yet described.